Compositions and methods for treatment of bone defects

ABSTRACT

The present invention relates to the field of treatment of bone defects. The invention provides a composition for treatment of bone defects as well as methods for treatment of such defects.

FIELD OF INVENTION

The present invention relates to the field of treatment of bone defects.The invention provides a composition for treatment of bone defects aswell as methods for treatment of such defects.

BACKGROUND OF INVENTION

Bone defects represent a common orthopaedic problem and can be asignificant challenge to treat. They can arise as a consequence ofdisturbances or failure of formation and/or differentiation (boneaplasias and dysplasias), trauma, destruction due to a pathologicalprocess (infection, inflammation, tumors and implant wear associatedosteolysis) or a surgical intervention or any combination of these.

The three principal factors beneficial for the treatment of any bonedefect regardless of its etiology, namely osteoconductive,osteoinductive and osteogenetic properties have been known for decades.In 2007, Giannudis et al added to these another vitally importantfactor, mechanical stability, coining the “diamond concept” of bonedefect reconstruction.

However, a single best method for optimal delivery of these fourproperties in any given situation with its own special circumstancesparticular to the respective individual case does not exist and has yetto be described.

Every year, many patients with benign bone tumors of greatly diverseetiology are treated. In addition, there is an increasing need forreconstruction of bone destruction secondary to metastatic bone disease(MBD) in a growing number of patients.

An optimal method for surgical reconstruction of metastatic bone defectshas yet to be described and in absence of better alternatives, suchdefects have traditionally been managed with either implantation ofallograft bone or bone cement (poly-methyl-methacrylate, PMMA). However,the bone remodeling capacity of allograft bone is limited, as this is anessentially dead material containing only a rather small amount ofosteoinductive factors and PMMA prevents any bone formation altogether.

Furthermore, recurrent disease, particularly in more aggressive tumortypes is not uncommon and hence the use of numerous adjuvant treatmentshave been described. Systemic administration of bisphosphonates, in thiscontext, is well known to strongly inhibit osteoclast mediated bonedestruction in primary and secondary bone tumors.

SUMMARY OF INVENTION

Drawbacks of conventional bone graft substitutes include delayed orabsent resorption, which may restrict or even block bone ingrowth andformation of new bone. In addition, some bone graft substitutes areassociated with unpredictable in situ maintenance of their solidstructure, which has led to treatment cases where adequate mechanicalstability of the bone reconstruction construct was not sufficientlymaintained and a stable osteoconductive scaffold was not achieved.

Accordingly there is a need for new and effective materials and methodsfor treatment of bone defects. Mechanical stability is necessary toallow an undisturbed biological healing process, physiologicalremodeling and regeneration of bone. In one embodiment the presentinvention provides a multi-phasic biodynamic scaffold useful forassembly and for building in situ to accommodate the individualcharacteristic of the individual bone defect.

The invention thus provides a kit-of-parts comprising a bonereconstruction composition comprising a biocompatible carrier and atleast one bioactive agent and a biocompatible matrix comprising solidsparticles and/or scaffolds of natural or synthetic origin. The solidparticles and/or scaffolds may contribute to mechanical stability.

In one aspect, the present disclosure also provides a compositioncomprising a bone reconstruction composition comprising a biocompatiblecarrier and at least one bioactive agent and a biocompatible matrixcomprising solids particlesand/or scaffolds of natural or syntheticorigin.

The components of the kit-of-parts may be used in a layered applicationand subsequent impaction achieves a firmly interdigitated threedimensionally solid composite structure. After curing of the compositefilling the interspaces, said structure optimally fits the individualdefect geometry and has vastly improved mechanical properties making itsubstantially more resistant to bending and shears forces. Thus,typically after 6 weeks and even more after 3 to 6 month from surgery,bone re-growth and remodeling is observed and there is no sign ofliquefaction of the composite, as can also be seen from the pictures inthe drawings.

Thus, the invention provides methods for treatment of a bone defect inan individual in need thereof, which method comprises

-   -   providing a bone reconstruction composition comprising a        biocompatible carrier (e.g. hydroxyapatite) and at least one        bioactive agent,    -   providing a biocompatible matrix comprising solids particles        and/or scaffolds of natural or synthetic origin    -   contacting the site of the bone defect in said individual with        alternating layers of said composition and biocompatible matrix    -   thereby treating said bone defect.

The disclosure also provides a method for treatment of a bone defect inan individual in need thereof, which method comprises

-   -   providing a bone reconstruction composition comprising        hydroxyapatite, a calcium salt and an antiresorptive agent,    -   providing a bone graft material comprising bone from a human or        non-human animal    -   contacting the site of bone defect in said individual with        alternating layers of said composition and said bone graft        material        thereby treating said bone defect.

The present disclosure also provides a method for bone reconstruction inan individual suffering from a bone defect, said method comprisingperforming the method according to any one of the preceding claims,thereby obtaining growth of healthy bone on the site of the bone defectin said individual.

The invention also provides kit-of-parts comprising

-   -   a bone reconstruction composition comprising an osteoconductive        carrier (e.g. hydroxyapatite) and at least one bioactive agent,

a biocompatible matrix comprising solids particles and/or scaffolds ofnatural or synthetic origin.

The invention also provides kit-of-parts comprising

-   -   a bone reconstruction composition comprising hydroxyapatite, a        calcium salt (e.g. calcium sulphate) and an antiresorptive agent        (e.g. a bisphosphonate),    -   a bone graft material comprising bone from a human or non-human        animal.

The invention also provides kits-of-parts for use in the treatment of abone defect in an individual in need thereof, wherein said kit-of-partscomprises or consists of

-   -   a bone reconstruction composition comprising a biocompatible        carrier (e.g. hydroxyapatite) and at least one bioactive agent,    -   a biocompatible matrix comprising solids particles of natural or        synthetic origin.

The invention also provides kits-of-parts for use in the treatment of abone defect in an individual in need thereof, wherein said kit-of-partscomprises or consists of

-   -   a bone reconstruction composition comprising hydroxyapatite, a        calcium salt and an antiresorptive agent,    -   a bone graft material comprising bone from a human or non-human        animal.

The invention furthermore provides bone reconstruction compositionscomprising a biocompatible carrier (e.g. hydroxyapatite), anantiresorptive agent (e.g. a bisphosphonate), and fosfomycin.

DESCRIPTION OF DRAWINGS

FIG. 1. 45 year old female with pathologic fracture through large giantcell tumor in the right distal femur, treated with open biopsy,curettage, burring, bone defect reconstruction with a combination ofBGS/ZA and cancellous allograft, augmented with lateral locking plateosteosynthesis. Post-op x-rays and CT scans show progressive remodelingmost prominent in the peripheral areas of the regenerate, as well assubstantial periosteal bone formation (arrows). a. pre-operative; b.Post-operative and c. 6 months.

FIG. 2. 38 year old female with symptomatic vascular malformation withsecondary aneurysmal bone in the left distal humerus, treated with openbiopsy, curettage, burring, bone defect reconstruction with acombination of BGS/ZA and cancellous allograft. Post-op x-rays and CTscans show homogenous, progressive remodeling of the entire regenerate.a. pre-operative; b. post-operative; c. 3 months and d. 6 months.

FIG. 3. 34 year old female with biopsy verified symptomatic PVNS in theleft proximal tibia, treated, curettage, burring, bone defectreconstruction with a combination of BGS/ZA and cancellous allograft.Post-op x-rays show progressive remodeling of the entire regenerate. A.pre-operative; b. post-operative and c.6 months.

FIG. 4. 26 year old female with symptomatic aneurysmal bone in the rightfemoral diaphysis verified by open biopsy and treated with curettage,burring, bone defect reconstruction with a combination of BGS/ZA andcancellous allograft and prophylactic fixation with a posterolaterallocking plate. Post-op x-rays show progressive remodeling and no sign oflocal recurrence. A. pre-operative; b. post-operative and c.6 months.

FIG. 5. 19 year old female with simple cyst and fracture in the rightfemur with minimal residual bone. The figures illustrate thereconstruction with locking plate and impaction grafting with allograft;BGS with zoledronic acid and demineralized bone matrix (DBM). 6 weekx-ray shows healing. a. pre-operative; b. post-operative and c. 6 weeks.

FIG. 6. 38 year old female with symptomatic vascular malformation withsecondary aneurysmal bone in the left distal humerus, treated with openbiopsy, curettage, burring, bone defect reconstruction with acombination of BGS/ZA and cancellous allograft. Post-op x-rays and CTscans show homogenous, progressive remodeling of the entire regenerateas well as substantial periosteal bone formation (arrows). a.pre-operative; b. post-operative; c. 3 months and d. 6 months.

FIG. 7. 35 year old female with benign bone lesion (probably old boneinfarct) in the proximal tibia, treated with open biopsy, curettage,burring, bone defect reconstruction with Cerament BVF (17 ml) only (A).Post op radiographs show progressive resorption (B) and liquefaction (C)and ultimately complete disappearance of the BGS without any sign ofbone formation on x-ray, MRI or CT scanning (D,E,F). a. post-operative;b, 6 weeks, c. 3 months and d, e and f. 6 months.

FIG. 8. 68 year old male with aseptic loosening of an intercalarydiaphyseal replacement in the right femur (A), treated with 2 stagerevision and intermediate implantation of an antibiotic cement spacer(B) and re-implantation of a new, cemented, silver coated intercalaryspacer and periprosthetic bone defect reconstruction (C). A composite ofRIA-autograft from the contralateral femur, Cerament G with ZA, DBM andallograft were applied postero-medially, while the remaining defectanterolaterally (arrow) was filled with BGS only. Radiographic follow upat 6 months shows substantial new bone formation in the area where thebioactive composite had been applied, whereas the area treated with theBGS only shows resorption of all material applied to this area and noevidence of bone formation (D). a. pre-operative, b. post-operative (1ststage), c. post-operative (2nd stage), d. 6 months.

FIG. 9. 8 year old boy with a large simple cyst in the left proximalfemur, treated with percutaneous injection of Cerament BVF (A).Immediate post-operative radiographs show almost complete filling of thecyst. 3 weeks post-operatively, the boy suffers a spontaneous fractureof the proximal femur though one of the BGS injection portals (B). Arevision operation is carried out, where parts of the BGS in the area ofthe fracture are removed, but the cranial two thirds appear structurallyintact and are left in place (B, arrow). After reconstruction of theremaining defect in the region of the fracture with Allograft, BGS+ZAand DBM, a pediatric LCP is applied to internally fix the fracture.Post-operative follow-up radiographs show sound progressiveconsolidation and remodeling of the fracture, while the BGS in thecranial parts of the cyst is progressively resorbed without any evidencebone formation in the femoral neck (arrows, C and D).

FIG. 10. 67 year old female with symptomatic giant cell tumor of bone inthe medial femoral condyle, treated with open biopsy, curettage,burring, bone defect reconstruction with a combination of BGS/ZA andcancellous allograft. Overall stability of the bone was deemedsufficient so that plate augmentation was not necessary. Immediate posta-p radiographs show complete tumor removal and defect filling inseveral ALIG layers (A). Follow up x-rays and CT scans show progressivehomogenous remodeling of the entire regenerate. a. pre-operative(inset), post-operative, b. 6 months, c and d. 12 months.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “biocompatible matrix” as used herein refers to a compositioncomprising solid particles and/or scaffolds of natural or syntheticorigin. Said biocompatible matrix is preferably mechanically stable, andthus may be capable of providing mechanical stability to bone. Saidparticles and/or scaffolds are biocompatible meaning that they cancoexist with living tissues without causing harm. Preferably saidparticles may slowly be resorbed by osteoclasts and subsequentlyreplaced by newly formed bone through osteoblastic activity, oralternatively, the particles may permanently osseointegrate. By“osseointegrate” it is understood that a direct structural andfunctional connection is formed between living bone and the surface ofthe particles and/or scaffolds, for example hydroxyapatite, allog raftbone or metal particles and/or scaffolds. The biocompatible matrix maybe resorbable, slowly resorbable or non-resorbable. The biocompatiblematrix may comprise or consist of a bone graft material. For example,the particles may comprise or consist of one or more selected from thegroup consisting of bone graft material (e.g. autograft and/or allograftbone graft material), synthetic metallic particles and/or scaffolds,synthetic non-metallic particles and/or scaffolds and any combinationthereof.

The term “biocompatible carrier” as used herein refers to a compositioncomprising materials of natural or synthetic origin (e.g. calcium salts,polymers, collagen gels) which can coexist with living tissues withoutcausing harm and are suitable vehicles for delivery of any bioactiveagent to a living tissue, for example to the site of a bone defect. Insome embodiments, the biocompatible carrier is an osteoconductivecarrier. In some embodiments, the biocompatible carrier comprises anosteoconductive material. The term “osteoconductive” as used hereinrefers to a material that can serve as a scaffold for new bone growththat is perpetuated by the native bone. Osteoblasts from the margin ofthe defect that is being treated can utilize the osteoconductivematerial as a framework upon which to spread and generate new bone. An“osteoconductive material” is usually a material that can be in powderform, but can become a paste when mixed with a solvent. The so-formedpaste can subsequently cure at physiological conditions e.g. in the siteof a bone defect.

The term “bone” as used herein refers to natural bone from any animal.Bones typically comprise cortical bone and cancellous bone. Bone mayalso comprise other types of tissue, which may be referred tocollectively as “soft tissue”. The soft tissues include marrow,endosteum, periosteum, nerves, blood vessels and cartilage.

The term “bone graft material” as used herein refers to a materialcomprising bone from a human or non-human animal. Preferably, bone graftmaterial consists exclusively of material from bone from a human ornon-human animal. Bone graft material is preferably divided inparticles.

The term “cancellous bone”, also known as trabecular bone or spongybone, as used herein refers to the bone composed of tiny lattice-shapedunits, typically located internal to the hard compact bone at themetaphyeal ends of the long bones.

The term “cure” as used herein in relation to the bone reconstructioncomposition, refers to hardening of the composition. After mixing allingredients of the bone reconstruction composition, the compositionstarts curing. The composition is considered “cured” if digitalcompression does not leave an impression.

The term “digital compression” as used herein refers to pushing hardwith a finger.

The term “paste” as used herein refers to a composition of a sufficienthigh viscosity to allow physical application to a defined space withoutlosing its shape. A composition is considered a “paste”, when theinterphase between the paste and surrounding air is not deformed when acontainer comprising the paste is being moved.

The term “individual” as used herein refers to a human or non-humananimal.

The term “resorbable” as used herein refers to a material that can betargeted by osteoclasts, dissolved, and subsequently replaced by newlyformed bone through osteoblastic activity. Resorbable materials can bepolymers, ceramics, or composites. Examples of resorbable materials arecalcium sulfate, calcium phosphate, hydroxyapatite, polymers, e.g.polylactcic acid, collagen foams or gels.

The term “treatment” as used herein may refer to any kind of treatment.The treatment may be a curative treatment, or it may also be anameliorating treatment and/or a treatment reducing the effects of thebone defect. The treatment may also be a treatment which delaysprogression of a bone defect, for example the treatment may reduce thegrowth of metastatic bone disease.

Bone Reconstruction Compositions and Kit-of-Parts Comprising Same

The present invention relates to kits-of-parts for bone reconstruction,wherein said kits typically comprise a bone reconstruction compositionand a biocompatible matrix. In particular, the biocompatible matrix maycomprise or consist of a bone graft material. Examples of usefulbiocompatible matrices are described below. The bone reconstructioncomposition may comprise a biocompatible carrier and at least onebioactive agent. In particular, the bone reconstruction composition maycomprise an osteoconductive material. Examples of useful biocompatiblecarriers and bioactive agents are provided below. In particular theosteoconductive material comprises or consists of hydroxyapatite andcalcium salts. In particular, the at least one bioactive agent may be anantiresoprtive agent, such as a bisphosphonate. Said bone reconstructioncomposition may also comprise two or more bioactive agents that can addfurther beneficial effects for bone reconstruction. Examples of suchbioactive agents are anti-microbial agents, antiresorptive agents,anti-tumor agents, bone growth-promoting osteoinductive and/orosteogenetic agents, and more as described in the section below“Bioactive agents”. Said bone reconstruction composition may alsocomprise a solvent. In some embodiments, the bone reconstructioncomposition comprises a bioactive agent which is in liquid form and canact as a solvent.

The invention also provides a bone reconstruction composition per se,which comprises a biocompatible carrier, an antiresorptive agent such asbisphosphonate, and typically also an anti-microbial agent such asfosfomycin and/or other bioactive agents. Examples of usefulbiocompatible carriers, bisphosphonates, anti-microbial agents and otherbioactive agents are provided below.

The bone reconstruction composition and the kit-of-parts are preferablyprepared in such a way that they can be used to repair bone defects invivo and stimulate bone growth in a subject affected by a bone defect.In particular, it is highly preferably that the bone reconstructioncomposition achieves a viscosity, which is sufficiently high so that itdoes not flow away from the site of the bone defect when added to saidsite. On the other hand, the bone reconstruction material shouldpreferably be moldable for a sufficient amount of time to allow foroptimal adaptation to the individual site and geometry during repair ofthe bone defect. Accordingly, it is preferred that the bonereconstruction composition may reach a beneficial degree of hardnesswithin 2 to 60 minutes from mixing. In particular it is preferred thatthe bone reconstruction composition cures in a time range of 2 to 120min, such as in the range of 2 to 60 minutes, for example in the rangeof 30 to 60 min. after all ingredients of said composition are mixed.

It is also preferred that the bone reconstruction composition hardens toa paste in a time range of 2 to 10 minutes, such as in a time range of 2to 5 minutes, such as in a time range of 2 to 3 minutes after allingredients of said composition are mixed.

It is also preferred that the bone reconstruction composition thickensto a paste in a time range of 2 to 10 minutes, such as in a time rangeof 2 to 5 minutes, such as in a time range of 2 to 3 minutes after allingredients of said composition are mixed.

The bone reconstruction compositions may also comprise bioactive agentsand therapeutic agents such as anti-microbial agents and/orantiresorptive agents, anti-tumor agents and growth-promoting agentsthat facilitate, enhance or protect the bone reconstruction. Thesebioactive agents and therapeutic agents may be released from thecomposition to the tissues around it in vivo in the treated individual.Examples of said bioactive agents are provided below.

In one embodiment, at least one bioactive agent is an antibiotic, asdescribed in the section “Antibiotic”. In another embodiment at leastone bioactive agent is an antiresorptive agent. For example, theantiresorptive agent may be one of the bisphosphonates described in thesection “Bisphosphonate”. In a further embodiment, the bonereconstruction composition may comprise a growth promoting agent, whichmay be any of the bioactive agents described in the section “Growthpromoting agent”.

Thus, the bone reconstruction composition may comprise any of thebioactive agents described herein below in the sections “Bioactiveagent”, “Bisphosphonate”, “Antimicrobial agents”, “Growth promotingagents” and “Other bioactive agents”. In particular, the bonereconstruction composition may comprise both an antibiotic and anantiresorptive agent. Alternatively, the bone reconstruction maycomprise only one of an antibiotic or an antiresorptive agent. Inaddition, the bone reconstruction composition may also comprise one ormore additional bioactive agents.

The main components comprised in the bone reconstruction composition mayhave any of the features described in the following sections“Biocompatible carrier”, “Bioactive agent”, “Hydroxyapatite”,“Bisphosphonate”, “Calcium salt”, “Antibiotic”, and “Growth factor”.

The kit-of-parts according to the invention typically comprises the bonereconstruction composition and a biocompatible matrix. The biocompatiblematrix comprises solid particles of natural or synthetic origin. Saidparticles may be metallic or non-metallic synthetic particles or bonegraft material, such as bone from a human or a non-human animal, and maybe any of the materials described herein below in the section“Biocompatible matrix”. Typically, the parts of the kit may be providedand stored separately.

Said kit-of-parts may be prepared for contacting the site of a bonedefect in an individual in need thereof with alternating layers of bonereconstruction composition and biocompatible matrix such as a bone graftmaterial in order to achieve the important mechanical stability of theresulting bone reconstruction construct to provide a mechanicallyundisturbed environment conductive to bone graft incorporation,remodeling and overall healing. Examples of useful methods forcontacting the site of a bone defect are described herein below in thesection “Treatment of bone defect”. Said biocompatible matrix may be anyof the materials described in the section below “Biocompatible matrix”.

The bone reconstruction composition may be replaced by the healthygrowing bone of the subject, said bone growing because of the beneficialeffects of the compositions and methods described in the presentinvention.

Examples of effects obtained when applying said kit-of-parts for bonereconstruction in an individual in need are described in the sectionbelow “Treatment of bone defect”.

Treatment of Bone Defects

The present invention describes methods for treatment of a bone defectin a subject, said treatment comprising providing a composition thatpromotes bone reconstruction, providing a biocompatible matrix e.g. abone graft material, and providing impacting a bone defect with saidcomposition and said biocompatible matrix in alternate layers. Thecomposition may comprise different components as described in thesection “Compositions for bone reconstruction”.

In a preferred embodiment of the present disclosure, a bone defect istreated using the method as disclosed herein and described in detail inthe section below “Method for bone reconstruction”.

In a preferred embodiment of the present invention, the treatmentcomprises local administration of a composition and a bone graftmaterial to a subject affected by a bone defect thereby allowing growthof healthy bone in place of a bone defect.

In one embodiment the method involves contacting the site of bone defectwith alternating layers of said bone reconstruction composition and saidbiocompatible matrix e.g. a bone graft material. This in-situ additivelayering may start with contacting the entire bone walls of the site ofbone defect with the bone reconstruction composition. This may ensurethat the biologically most active substances are delivered as close aspossible to the target zone of initial remodeling and potential residualpathology. Said bone reconstruction composition is typically deliveredin the form of a paste. After addition of said bone reconstructioncomposition the biocompatible matrix may be added.

In some embodiments of the present disclosure, the bone reconstructioncomposition and the biocompatible matrix e.g. a bone graft material areapplied to the bone defect by injection and/or moulding.

Thus, the method may comprise the steps of:

-   -   a) contacting the bone walls of the site of the bone defect to        be treated with the bone reconstruction composition of the        invention, wherein the bone reconstruction composition        preferably is in the form of a paste; and    -   b) contacting the bone reconstruction composition with a layer        of said biocompatible matrix;    -   c) contacting the biocompatible matrix with a layer of said bone        reconstruction composition;    -   d) optionally repeating steps b) and c) 1 to 20 times, such as 1        to 10 times, for example 1 to 5 times, such as 1 to 3 times        depending on the size of the bone defect, so that or few layers        are used to fill a small bone defect, whereas several layers are        used to fill a large bone defect.

Before step a) the method may comprise contacting the site of the bonedefect with one or more active compounds, e.g. with an antiresorptiveagent, such as a bisphosphonate, with an antimicrobial agent such as anantibiotic and/or with a growth promoting agent, such as a BMP.

In some embodiments of the present disclosure, Zoledronic acid or apharmaceutically acceptable salt thereof is administered locally to thesite of the bone defect prior to performing step a).

In one embodiment of the present invention, bone density measurementswith dual energy x/ray absorptiometry (DEXA) are performed before andafter treatment to assess the state of the bone. In a preferredembodiment, the values do not change significantly in the time followingtreatment with the kit-of-parts and methods according to the presentinvention. Exemplary values before treatment are at least 1.5 g/cm² andnot higher than 3 g/cm². In an individual treated with traditionalmethods, said values would typically drop to at least 0.1 g/cm² and nothigher than 1 g/cm² after treatment. In an individual treated with thekit-of-parts and methods according to the present invention, said valueswould remain constant and thus be at least 1.5 g/cm² and not higher than3 g/cm² after at least 6 weeks, such as at least 12 weeks aftertreatment, indicating that bone has replaced the composition.

The methods for treatment of a bone defect in a subject disclosed hereinmay be supplemented by the use of appropriate internal or external bonefixation devices to establish or protect mechanical integrity of theentire bone in which the defect is treated. As these foreign bodiesrepresent a risk for microbial colonization and biofilm formation,therapeutic- and/or preventive antimicrobial biofilm agent applicationto the surface of the implant may be needed. The bioactive agentsdescribed in the sections “Bioactive agents”, “Antibiotics” and “Otherbioactive agents” can be used for this purpose. Said agents may beapplied as preliminary surface treatment (e.g. surface coating withantimicrobial agents such as silver, copper, antibiotics or others).Said agents may also be used for in-situ surface modification of thesurface of the implant e.g. with antibiotic- and/or antibiofilm agents.

In some embodiments of the present disclosure, the biocompatible carriercomprises at least one bioactive agent, wherein said bioactive agentprevents and/or treats microbial colonization and biofilm formation ofthe particles of the biocompatible matrix.

In some embodiments of the present disclosure, the biocompatible carriercomprises at least one bioactive agent, wherein said bioactive agentprevents and/or treats microbial colonization and biofilm formation ofany internal or external bone fixation device used for treating a bonedefect.

Bone Defect

The present invention describes methods, kits-of-parts and bonereconstruction compositions that are useful for treatment of a varietyof bone defects in a subject. For example said individual may be anindividual suffering from cancerous bone diseases, for examplemetastatic bone lesions; primary bone cancer, for example osteosarcoma;benign bone tumors, for example giant cell tumor of bone; inflammationor infection of the bones; and one or more bone fractures, for examplepathologic fractures, and thus, said bone defect may be any of theaforementioned or caused by any of the aforementioned conditions.

The bone reconstruction compositions, the kits-of-parts as well as themethods can also be used to reconstruct a gap, a bone void, or apre-existing bone cavity. Accordingly, the bone defect may be a partialor complete structural gap, a so-called segmental bone defect, or a bonevoid or a pre-existing bone cavity, so-called cavitary bone defect.

The bone reconstruction compositions, the kits-of-parts as well as themethods can be used in a wide variety of applications, such as in thetreatment of bone defects associated with trauma and fracture healing,prosthetic implants, and implants of foreign materials in othersituations and bone loss due to congenital, infective or iatrogeniccauses. Such situations may comprise filling a gap or a bone void or apre-existing bone cavity, such as fractures, osteotomy, for theattachment of prostheses or other foreign material, for prostheticrevision surgery, for plastic surgery, for reconstruction surgery, orfor cosmetic surgery.

The bone reconstruction compositions, the kits-of-parts as well as themethods are suitable for local use in tooth pockets and/or bifurcaturesto treat periodontitis or to be combined with other treatment optionsfor periodontitis included therein, such as supportive matrix proteinsor locally acting growth inducing factors.

Likewise, they can be used together with collagen membranes or othersupports, which are of importance for the growth of supportive tissues.

The bone reconstruction compositions, the kits-of-parts as well as themethods can be used for repairing osteochondral defects as well asfractures or bone defects involving a joint.

The bone reconstruction compositions, the kits-of-parts as well as themethods can be used to fill skeletal defects caused by the removal oforthopedic devices, which are utilized for internal or external fixationof fractures, for example screws and pin tracts. It is in such occasionspreferred that antibiotics are included in the composition as additives.They can also be used for filling of a bone cavity or replacing bonelost during surgical removal of a tumor.

The bone reconstruction compositions, the kits-of-parts as well as themethods can be used for local treatment of infections or infestations inthe musculoskeletal system, such as osteomyelitis caused by e.g.bacteria or fungi. A combined osteoplastic and local antimicrobialtreatment and a prophylaxis of skeletal infection can thus be obtained,e.g. in sternotomies, prosthetic implants, reconstructive surgery,trauma surgery, cancer surgery, cosmetic surgery, and oro-maxillo-facialsurgery.

The bone reconstruction compositions, the kits-of-parts as well as themethods can also be used for local treatment with cytostatic oranti-tumor agents, such as in musculoskeletal tumors, e. g. fortreatment of metastases in bone, e.g. in vertebrae. The metastases maybe from any primary cancer, such as breast or prostatic cancers. Theprimary cancer may be a cancer demanding treatment with a supportivematerial that concomitantly gives a possibility for local treatment withanti-tumor agents. Furthermore, it can be used locally together withagents that enhance the clinical effects of irradiation in diseases,such as tumor diseases. A list of relevant anti-tumor agents that can beincorporated in the bone reconstruction composition as bioactive agentscan be found on https://www.cancer.gov/about-cancer/treatment/drugs.

In one embodiment the bone defect is caused by an inflammation and/or byan infection of the bone.

In one preferred embodiment of the invention the bone defect is a bonetumor, such as giant cell tumor of bone and/or the bone defect isassociated with or caused by a bone tumor, such as giant cell tumor ofbone.

In another preferred embodiment of the invention the bone defect ismetastatic bone disease. Many primary tumors metastasize to otherlocations in the body, for example bone. Cancer which has metastasizedto bone may be referred to as “metastatic bone disease”. A metastasis tothe bone may cause bone cavities. For example, metastases may be removedfrom the bone by surgery leaving a bone cavity. Bone cavities caused byor associated with metastases to the bone may be filled using the bonereconstruction compositions, the kits-of-parts and/or the methods of thepresent invention.

Biocompatible Carrier

The bone reconstruction compositions described by the present disclosurecomprise a biocompatible carrier.

Said biocompatible carrier may be a controlled releasing polymer matrix,for example comprising a synthetic polymer, such a synthetic polymerwith pendant-functionalized diols, polyethylene glycol, polyacrylicacid, polyesters and their co-plymers e.g. polycaprolactone,polyanhydrides e.g. polyanhydrides based on sebacic acid (SA),p-(carboxyphenoxy)propane (CPP), p-(carboxyphenoxy)hexane (CHP) andtheir copolymers, polyamides, polyorthoesters (POE), e.g. POE I, POE II,POE III and POE IV; recombinant proteins-based polymers, in-situ forminghydrogels (such as PHEMA, and copolymers of PVA or PEG withacrylamides).

Said biocompatible carrier may be a controlled releasing polymer matrix,for example comprising a natural polymer e.g. proteins such as collagen,gelatin and fibrin; polysaccharides such as hyaluronic acid andalginate; cellulose derivatives, chitosan, polysaccharide-based polymersand natural protein-based polymers.

Said biocompatible carrier comprising a releasing polymer matrix maycomprise polymers as described above in pure form or as part of drugdelivery systems such as nanoparticles and microparticles, dendrimers,nano- and micro-spheres, capsosomes and micelles.

In some embodiments, the biocompatible carrier comprised in the bonereconstruction composition comprises an osteoconductive resorbablematerial.

The biocompatible carrier is typically used as a carrier for one or morebioactive agents. The bioactive agents may further enhance the growth ofthe natural bone and/or have a therapeutic effect for treatment of thebone defect or a disease associated therewith. In some embodiments, thebiocompatible carrier comprises an osteoconductive material and so itfavors growth of natural bone via the action of osteoblasts andosteoclasts. In other embodiments, the biocompatible carrier does notcomprise an osteoconductive material.

In preferred embodiments of the present disclosure, the biocompatiblecarrier comprises calcium salts and/or hydroxyapatite, as describedbelow. In further embodiments of the present disclosure, thebiocompatible carrier comprises hydroxyapatite and polymers. It ispreferred that the biocompatible carrier comprises both calcium salt(s)and hydroxyapatite, which may be any of the calcium salts andhydroxyapatites described below.

Calcium Salt

The biocompatible carrier of the present disclosure may comprise calciumsalts. Said calcium salt may be any useful calcium salt. For example thecalcium salt may be selected from the group consisting of calciumsulfate and calcium phosphate.

In some embodiments of the invention the biocompatible carrier comprisesmore than one calcium salt, and thus the calcium salt may be a mixtureof at least two different calcium salts. Thus, the calcium salt may forexample comprise at least one calcium phosphate component and at leastone calcium sulfate component.

In other embodiments the calcium salt comprises only one calcium salt.

Calcium sulfate may particularly be hardened calcium sulfate, such ashardened calcium sulfate that has a diameter which is less than 100 μm.

In one embodiment of the present invention, calcium sulfate represent upto 100% of the calcium salts. In another embodiment, the particulatehardened calcium sulfate is calcium sulfate dihydrate (gypsum).

In one embodiment of the present disclosure, the biocompatible carrierdoes not comprise calcium sulfate.

Hydroxyapatite

The biocompatible carrier of the present disclosure may comprise thecalcium-rich mineral hydroxyapatite (HA). HA is a naturally occurringmineral and has the general formula Ca₅(PO₄)₃(OH), usually writtenCa₁₀(PO₄)₆(OH)₂ because its crystal unit is a dimer. Up to 50% by volumeand 7% by weight of human bone is a modified form of HA and HA is alsoknown as bone mineral.

When preparing the bone reconstruction composition, then typically theHA is mixed with the calcium salt. The ratio between the calcium saltand HA is typically in the following ranges:

-   -   30 to 50 parts HA: 50 to 70 parts calcium salt, e.g. calcium        sulfate, calcium phosphate or other salts    -   35 to 45 parts HA: 55 to 65 parts calcium salt, e.g. calcium        sulfate, calcium phosphate or other salts    -   40 parts HA: 60 parts calcium salt, e.g. calcium sulfate,        calcium phosphate or other salts.

The bone reconstruction composition may comprise at least 40% of saidmixture of HA and calcium salt, for example at least 50% of said mixtureof HA and calcium salt, such as in the range of 40 to 70%, for examplein the range of 50 to 70%, such as in the range of 50 to 60% of saidmixture of HA and calcium salt.

Bioactive Agents

The bone reconstruction compositions described by the present disclosurecomprise at least one bioactive agent. The at least one bioactive agentis usually comprised in the biocompatible carrier.

In some embodiments of the present disclosure, a bioactive agent isapplied directly to the solid components of the biocompatible matrix,for example silver or hydroxyapatite can be used to coat trabeculartitanium spheres, or antibiotics, anti-biofilm and antiresorptive agentscan be adsorbed onto purified allograft bone.

In some embodiments of the present disclosure the biocompatible carriercomprises one or more bioactive agents selected from the groupconsisting of antiresorptive agents, antibiotics, antibacterial agents,anti-biofilm agents, antiadhesive agents, antibodies, growth anddifferentiation factors, cytokines, bone morphogenetic proteins (BMP)antagonist inhibitors, signaling proteins, human cell suspensions,solvents, anti-tumor agents and radioactive material or combinationsthereof.

The bone reconstruction composition of the present disclosure comprises,in some embodiments, two or more bioactive agents, wherein any one ofthe two or more bioactive agent is any one of the bioactive agentsdescribed herein.

In some embodiments of the present disclosure, the bone reconstructioncomposition is in the form of a paste and comprises two or morebioactive agents, wherein at least one of the bioactive agentsaccelerates curing of the paste and wherein at least one of thebioactive agents decelerates curing of the paste.

Most additives and bioactive agents have a decelerating effect curing ofthe paste of the composition and a destabilizing effect on themechanical properties of the biocompatible carrier in-situ. Therefore,in some embodiments an agent that accelerates curing of the compositionmay be added. Said agent that accelerates curing of the bonereconstruction composition and improves its stability may be added bytitration.

In a preferred embodiment of the present disclosure, the bioactive agentthat accelerates curing of the composition is an antibiotic, for examplefosfomycin or a pharmaceutically acceptable salt thereof.

In a preferred embodiment of the present disclosure, the bioactive agentthat accelerates curing of the composition is fosfomycin or apharmaceutically acceptable salt thereof and said fosfomycin or apharmaceutically acceptable salt thereof is dissolved in a solvent.

In a further preferred embodiment of the present disclosure, thebioactive agent that decelerates curing of the composition is abisphosphonate, for example a bisphosphopnate selected from a groupconsisting of Zoledronic acid, Pamidronic acid, Neridronic acid,Olpadronic acid, Alendronic acid, Ibandronic acid, Risedronic acid,pharmaceutically acceptable salts of any of the aforementioned andcombinations of any of the aforementioned. For example, the bonereconstruction composition may comprise a bisphosphonate in an amountcomprised between 0.08 and 0.2 g/ml, so 10 ml of compositions maycomprise between 0.8 and 2 g of bisphosphonate.

Bisphosphonate

In some embodiments of the present disclosure, the bone reconstructioncompositions comprise a bisphosphonate. The term “bisphosphonate” asused herein refers to a class of medicaments that prevent the loss ofbone mass. This class of medicaments is also referred to asantiresorptive agents, as they slow or block the resorption of bone.

In a preferred embodiment of the present invention, the antiresorptiveagent used is a bisphosphonate. In a further embodiment, theantiresorptive agent is a compound having antiresorptive activity butnot the structure of a bisphosphonate, for example a monoclonal antibodysuch as Denosumab.

Preferably bisphosphonates to be used with the present invention havethe general structure of formula (I):

The bisphosphonates to be used with the present invention are preferablycompounds of formula (I), which are specific inhibitors of osteoclasts.By inhibiting osteoclasts, the bisphosphonates inhibit bone loss.

Typically, R′ may be a small moiety, for example R′ may be selected fromthe group consisting of —H, —OH and halogen. In a preferred embodimentR′ is —OH, which enhances binding to hydroxyapatite.

R″ may be a larger moiety although it is also comprised in the inventionthat R″ may be a small moiety such as halogen or methyl. Preferably,however R″ is —C₁₋₆-alkyl-X, wherein X is selected from the groupconsisting of cyclic, aryl, heteroaryl and amine. In particular, X maybe amine or a mono- or bicyclic heteroaryl, wherein at least oneheteroatom of said heteroaryl is nitrogen. Said amine may be a primaryamine, for example —NH₂, a secondary amine or a tertiary amine. Saidtertiary amine may be —N(R₁)(R₂), wherein R₁ and R₂ independently areC₁₋₆-alkyl. Said mono- or bicyclic heteroaryl, wherein at least oneheteroatom of said heteroaryl is nitrogen may for example be imidazole,pyridine or pyrrolidine.

In one embodiment of the invention the bisphosphonate is a nitrogencontaining bisphosphonate. Thus, the bisphosphonate may be abisphosphonate of formula (I), wherein at least one of R′ and R″contains nitrogen.

In one embodiment the bisphosphonate is selected from the groupconsisting of Zoledronic acid, Pamidronic acid, Neridronic acid,Olpadronic acid, Alendronic acid, Ibandronic acid, Risedronic acid andpharmaceutically acceptable salts thereof. The bone reconstructioncompositions may also comprise more than one bisphosphonate, for examplethe bone reconstruction compositions may comprise a combination of twoor more of the aforementioned bisphosphonates.

In another embodiment the bisphosphonate is a nitrogen containingbisphosphonate, for example selected from the group consisting ofZoledronic acid, Alendronic acid, Risedronic acid and pharmaceuticallyacceptable salts thereof.

In a preferred embodiment of the invention, the bisphosphonate isZoledronic acid (ZA) or a pharmaceutically acceptable salt thereof. Inparticular the bisphosphonate may be Zoledronic acid. Zoledronic acidmay also be referred to as zoledronate. Zoledronic acid is a compound ofthe formula (II):

Zoledronate has multiple anti-tumor effects, including direct inductionof cell death in various types of tumor, such as lung-, breast, prostateand kidney cancer, myeloma and giant cell tumor of bone.

The bisphosphonate may be present within the composition in any suitableamount. For example, the bone reconstruction composition may comprise inthe range of 0.1 to 1.5 mg/ml, such as in the range of 0.2 to 1 mg/ml,for example in the range of 0.2 to 0.0 mg/ml, such as in the range of0.3 to 0.8 mg/ml of said bisphosphonate. In one embodiment the bonereconstruction composition comprises approximately 0.3 mg/ml ofbisphosphonate. In one embodiment the bone reconstruction compositioncomprises approximately 0.55 mg/ml. In one embodiment the bonereconstruction composition comprises approximately 0.80 mg/ml. ml refersto the final volume of the bone reconstruction composition and the term“approximately” as used herein refers to +/−10%, such as +/−5%, forexample +/−1%.

In a preferred embodiment of the present disclosure, 10 ml of bonereconstructing composition comprises between 1 and 2.5 ml of saidZoledronic acid or pharmaceutically acceptable salt thereof, such asbetween 1 and 1.5 ml, such as between 1 and 2 ml, such as between 1. 5and 2 ml, such as between 1.5 and 2.5 ml, such as between 2 and 2.5 ml fsaid Zoledronic acid or pharmaceutically acceptable salt thereof.

In a preferred embodiment of the present disclosure, 10 ml of bonereconstructing composition comprises between 0.2 and 2 g of saidZoledronic acid or pharmaceutically acceptable salt thereof, such asbetween 0.2 and 1.5 g, such as between 0.2 and 1 g, such as between 0.2and 0.5 g, such as between 0.5 and 2 g, such as between 0.5 and 1.5 g ofsaid Zoledronic acid or pharmaceutically acceptable salt thereof.

Antimicrobial Agents

The compositions described by the present invention may compriseanti-microbial agents, such as antibiotics. Said agents may be releasedfrom the composition in vivo and therefore prevent, limit or treatinfection in the treated subject.

Such compounds include natural antibiotics as well as othersemisynthetic and synthetic antibacterial or bacteriostatic compounds,which are acting against pathogenic and/or infectious microorganisms,e.g. staphylococci. Non-limiting examples of antibiotics which can beused with the invention includes fosfomycin, gentamicin,tetracycline-HCl, vancomycin, Amikacin, Gentamicin, Kanamycin, Neomycin,Netilmicin,Tobramycin, Paromomycin, Streptomycin, Spectinomycin,Geldanamycin, Herbimycin, Rifaximin, Loracarbef, Ertapenem, Doripenem,Imipenem/Cilastatin, Meropenem, Cefadroxil, Cefazolin, Cefalotin orCefalothin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil,Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefotaxime, Cefpodoxime,Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cephalosporins(Fourth generation), Cefepime, Maxipime, Cephalosporins (Fifthgeneration), Ceftaroline fosamil, Ceftobiprole, Clycopeptides,Teicoplanin, Telavancin, Dalbavancin, Oritavancin, Lincosamides,Clindamycin, Lincomycin, Lipopeptide, Daptomycin, Macrolides,Azithromycin, Clarithromycin, Dirithromycin, Erythromycin,Roxithromycin, Troleandomycin, Telithromycin, Spiramycin, Monobactams,Aztreonam, Nitrofurans, Furazolidone, Nitrofurantoin, xazolidinones(Bs),Linezolid, Posizolid, Radezolid, Torezolid, Penicillins, Amoxicillin,Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin,Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin,Penicillin G, Penicillin V, Piperacillin, Penicillin G, Temocillin,Ticarcillin, Amoxicillin/clavulanate, Ampicillin/sulbactam,Piperacillin/tazobactam, Ticarcillin/clavulanate, Polypeptides,Bacitracin, Colistin, Polymyxin B, Quinolones/Fluoroquinolone,Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin,Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin,Sulfonamides(Bs), Mafenide, Sulfacetamide, Sulfadiazine, Silversulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole,Sulfanilimide (archaic), Sulfasalazine, Sulfisoxazole,Trimethoprim-Sulfamethoxazole (Co-trimoxazole) (TMP-SMX),Sulfonamidochrysoidine (archaic), Tetracyclines(Bs), Demeclocycline,Doxycycline, Minocycline, Oxytetracycline, Tetracycline, Clofazimine,Dapsone, Capreomycin, Cycloserine, Ethambutol (Bs), Ethionamide,Isoniazid, Pyrazinamide, Rifampicin (Rifampin in US), Rifabutin,Rifapentine, Streptomycin, Arsphenamine, Chloramphenicol(Bs),Fosfomycin, Fusidic acid, Metronidazole, Mupirocin, Platensimycin,Quinupristin/Dalfopristin, Thiamphenicol, Tigecycline(Bs), Tinidazole,or Trimethoprim(Bs).

In a preferred embodiment of the present disclosure, the antibiotic isselected from the group consisting of fosfomycin, gentamicin,tetracycline, vancomycin, tobramycin, gentamycin, cephalosporin and acombination thereof.

In a preferred embodiment of the present disclosure, the at least onebioactive agent comprises or consists of fosfomycin or apharmaceutically acceptable salt thereof.

For example, the bone reconstruction composition comprises at least 80mg/ml of fosfomycin, such as at least 97 mg/ml, such as at least 110mg/ml, and up to 200 mg/ml, such as up to 175 mg/ml of fosfomycin orpharmaceutically acceptable salt thereof, where ml refers to the finalvolume of the composition.

In a preferred embodiment of the present disclosure, the biocompatiblecarrier comprises an osteoconductive material such as hydroxyapatite andat least one bioactive agent, such as fosfomycin or a pharmaceuticallyacceptable salt thereof.

In a further embodiment of the present disclosure, the biocompatiblecarrier comprises an osteoconductive material such as hydroxyapatite anda bisphosphonate, such as Zoledronic acid, and at least anotherbioactive agent, such as fosfomycin or a pharmaceutically acceptablesalt thereof.

In fact, fosfomycin accelerates curing of a composition comprisinghydroxyapatite and may improve mechanical stability of said compositionin-situ.

The bone reconstruction composition may also comprise a cytostaticagent. Cytostatic agents, such as Bendamustine, Busulfan, Carmustine,Chlorambucil, Cyclophosphamide, Dacarbazine, Ifosfamide, Melphalan,Procarbazine, Streptozocin and Temozolomide or derivatives thereof maythus also be comprised in the bone reconstruction composition. The bonereconstruction composition can in a similar way comprise an antiviralcompound, an antifungal compound, a tuberculostatic or tuberculocidalcompound or an antiparasite compound or a genetically altered variant ofthe aforementioned for the purpose of targeted antitumor treatment.

Another additive, which may be included in the bone reconstructioncomposition, is a non-ionic X-ray contrast agent.

The antibiotic agent may be present within the composition in anysuitable amount. For example, at least 80 mg/ml, such as at least 97mg/ml, such as at least 110 mg/ml, and up to 200 mg/ml, such as up to175 mg/ml of said antibiotic or antibiotic mixture, where ml refers tothe final volume of the composition.

Different types of antimicrobial/anti-biofilm agents may be comprised inthe bone reconstruction composition to complement each other and deliversynergistic antimicrobial effects to the site of the bone defect. Saidbioactive agents will spread into the biocompatible matrix e.g. the bonegraft material as well as into the area around the bone defect, e.g.into local hematoma and/or seroma by way of elution and diffusion fromthe composite.

A list of anti-microbial agents suitable for use in combination withbiocompatible materials, such as for preventing or treating colonizationof said materials by microorganisms can be found in Campoccia et al.(2013. A review of the biomaterials technologies for infection-resistantsurfaces Biomaterials, 34(34): 8533-8554).

In one embodiment of the present disclosure, the at least one bioactiveagent comprises or consists of a biofilm preventing, dispersing ordisrupting agent, also referred to as anti-biofilm agent. Severalcompounds act as anti-biofilm agent, for example non-antibioticcompounds with innate antibacterial properties, such as silver andcopper; N-acetylcysteine; nitric oxide (NO)-releasing coatings withdiazeniumdiolates or calcium peroxide; bioactive antibacterial coatings;antimicrobials able to bypass the biofilm barrier, such as Daptomycinor, Minocycline; peptides such as human β-defensin-3; cyclic di-GMPmodulating proteins; the transcriptional regulatory protein MucR;nanostructured compounds such as silver or chitosan; enzymes that lysecertain elements of the biofilm e.g. dispersin B or proteinase K. Alsocytotoxic agents, which are described in detailed above, have been foundto be successful in removing biofilms from implant surfaces. Forexample, citric acid was found to be successful in eliminating biofilmsfrom titanium surfaces. A list of other agents that inhibit biofilmformation is found in Rabin N et al. (Rabin N, Zheng Y, Opoku-Temeng C,Du Y, Bonsu E and Sintim H O, Future Medicinal Chemistry, 2015 ,7(5):647-671).

Growth Promoting Agents

The bone reconstruction compositions described by the present inventionmay comprise agents that stimulate and/or accelerate bone formation(also referred to as “growth promoting agents”), such as osteoinductiveagents, growth factors and hormones. Specific signaling molecules,growth factors and derivatives thereof, which are locally acting arepreferred. Said agents may be released from the composition in vivo.

Appropriate stem cell differentiation and subsequent bone formationrequires concerted generation, conduction and regulation of a range ofspecific signaling molecules, including various enzymes, growth factorsand hormones. The biocompatible carrier of the present disclosure isalso an effective delivery vehicle for bioactive agents such as growthfactors and antiresorptive agents.

It is preferred to use autologous signaling molecules and growthfactors, which are effective in connection with bone, tendon orcartilage. Thus, the bone reconstruction composition may comprise atleast one growth or differentiation factor selected from the groupconsisting of bone morphogenic proteins (BMPs), members of theTGF-superfamily, platelet-derived growth factor (PDGF), fibroblastgrowth factors (FGFs), insulin-like growth factors (IGFs),metalloproteinases, vascular endothelial growth factor (VEGFs A, B, C orD), angioprotein 1 and 2, and combinations thereof. These endogenouslyproduced growth factors are used as an additive either as singleentities or combined in a growth factor mixture in order to acceleratebone growth. Thus, it is preferred that an endogenously producedbioactive molecule is used as a substance that induces bone formation.

In one embodiment of the disclosure the growth promoting orosteoinductive agent is a bone morphogenic protein (BMP, for example thegrowth promoting agent may be selected from the group consisting ofBMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP15 anda combination thereof. Preferably, said BMPs are BMPs of the samespecies to be treated with the bone reconstruction composition. Thus,the BMP may be human BMP, for example the growth promoting agent may beselected from the group consisting of human BMP1, human BMP2, humanBMP3, human BMP4, human BMP5, human BMP6, human BMP7, human BMP8a, humanBMP8b, human BMP10, human BMP15 and a combination thereof. The term“human BMP” as used herein refers to wild type human BMP.

Examples of other growth promoting agents are parathyroid hormones andderivatives thereof, estrogens, progesterone, androgens, testosterones,calcitonin, somatomedin, and oxytocin, preferably also autologous, butthey can also be produced according to procedures known within the art.

Other Bioactive Agents

The bone reconstruction compositions described by the present disclosurecomprise at least one bioactive agent e.g. a biologically and/ortherapeutically active agent, also referred to as bioactive agent. Inaddition to bisphosphonates, anti-microbial agents and growth promotingagents, other bioactive agents may be comprised in the composition, asdescribed here below.

In one embodiment of the present disclosure, the at least one bioactiveagent comprises or consists of a BMP antagonist inhibitors selected fromthe group consisting of noggin, chordin, sclerostin, follostatin.

In one embodiment of the present disclosure, the at least one bioactiveagent comprises or consists of a BMP antagonist inhibitors selected fromthe group consisting of members of the DAN family, for examplesclerostin, members of the twisted gastrulation family andnoggin/chordin.

In one embodiment of the present disclosure, the at least one bioactiveagent comprises or consists of a signaling protein, for example asignaling protein selected from the group consisting of Wnt proteins,lymphoid enhancer-binding factor 1 (Lef1), β-catenin, parathyroidhormone-related protein (PTHrP), colony stimulating factors, Indianhedgehog homolog (IHH), Hypoxia-inducible factor 1-alpha (HIF-α) andcombinations thereof.

Healing and generation of new mineralized tissue in bone defects, theso-called osteogenesis involves active participation of various types ofcells, including osteoblasts, chondroblasts, osteocytes and osteoclasts.Bone formation is the result of successful recruitment anddifferentiation of locally available and/or implanted pluripotentprogenitor cells into mature bone matrix producing cells at the site ofthe lesion. Suspensions of pluripotent mesenchymal stem cells (MSC's)and/or platelet rich plasma (PRP) can be harvested form a variety ofsources (e.g. blood, bone marrow and/or adipose tissue), incorporatedinto an appropriate biocompatible carrier, preferably comprising anosteoconductive material, and thus delivered to the site of a bonedefect as part of the method disclosed herein.

Similarly, other types of cells, such as for example tumor infiltratinglymphocytes could be incorporated in a similar fashion into a surgicallytreated bone tumor site, to allow local delivery of cells for specificpurposes, such as the killing of tumor cells, which may enhance localrecurrence rates.

Therefore, in one embodiment of the present disclosure, the at least onebioactive agent comprises or consists of a human cell suspension, forexample a human cell suspension comprising or consisting of mesenchymalstem cells and/or lymphocytes and/or platelet depleted plasma and/orlipoaspirate of autogenic or allogenic origin such as the stromalvascular fraction.

In some embodiments of the present disclosure, cell suspensions areadded to or substitute part of the solvent comprised in thebiocompatible carrier.

In cases of treatment of metastatic bone lesions or pathologicfractures, placement of pellets or seeds in the biocompatible carrierwould permit localized internal radiation therapy of eventual residualtumor tissue. The internal radiation therapy delivered from radiationsources placed inside or on the body is termed brachytherapy. Inbrachytherapy, radioactive isotopes are sealed in tiny pellets or“seeds” which can be delivered to patients using delivery devices, e.g.an appropriate biocompatible carrier, such as a carrier comprising anosteoconductive material. As the isotopes decay naturally, they give offradiation that damage nearby cancer cells. If left in place, after a fewweeks or months, depending on the isotope, the isotopes decay completelyand no longer give off radiation. The seeds will not cause harm if theyare left in the body. Brachytherapy may be able to deliver higher dosesof radiation to some sites than external-beam radiation therapy whilecausing less damage to normal tissue.

The seeds can be resorbable or non-resorbable.

The placement of brachytherapy sources can be temporary or permanent.These particles or seeds may be also embedded into the biocompatiblematrix.

In some embodiments, these solid particles are embedded into thebiocompatible carrier.

Therefore, in one embodiment of the present disclosure, the at least onebioactive agent comprises or consists of a radioactive material, forexample a radioactive material selected from the group consisting ofCesium-131 or Cesium-137 or Cobalt-60 or Iridium-192 or Iodine-125 orPalladium-103 or Ruthenium-106 or Radium-226 and combinations thereof.

In one embodiment of the present disclosure, the at least one bioactiveagent comprises or consists of a cytokine, for example a cytokineselected from the group consisting of interleukin-1 (IL-1) orinterleukin-6 (IL-6) or tumor necrosis factor alpha (TNF-alpha) orreceptor activator of nuclear factor kappa-B ligand (RANKL) orcore-binding factor alpha 1 (cbfa1) or osteogenin or SMADs proteins 1-8or osteogenic growth peptide (OGP) and combinations thereof.

In one embodiment of the present disclosure, the biocompatible carriercomprises a solvent, for example a solvent selected from the groupconsisting of water, simple sugars such as for example glucose,fructose, mannitol, a contrast agent such as iohexol, platelet depletedplasma, lipoaspirate, fosfomycin, vancomycin or gentamycin andcombinations thereof.

In one embodiment of the present disclosure, the at least one bioactiveagent comprises or consists of an antiadhesive agent, for examplemonomeric trimethylsilane (TMS) or nonionic surfactants, e.g PluronicF127.

In one embodiment of the present disclosure, the at least one bioactiveagent may be an antimetabolite, a chemical that inhibits the use of ametabolite which is part of the normal metabolidm of the subject. Theantimetabolite is selected from the group consisting of Asparaginase,Capecitabine, Cytarabine, 5-Fluoro Uracil, Fludarabine, Gemcitabine,Methotrexate, Pemetrexed and Raltitrexed.

In another embodiment of the present disclosure, the at least onebioactive agent may be an anti-tumour antibiotic selected from a groupcomposed of Actinomycin D/Dactinomycin, Bleomycin, Daunorubicin,Doxorubicin, Doxorubicin (pegylated liposomal), Epirubici, Idarubicin,Mitomycin and Mitoxantrone. In another embodiment of the presentinvention, bioactive agents, e.g. biologically and/or therapeuticallyactive agents, may be plant alkaloids/microtubule inhibitors selectedfrom a group composed of Etoposide, Docetaxel, Irinotecan, Paclitaxel,Topotecan, Vinblastine, Vincristine and Vinorelbine.

In another embodiment of the present disclosure, the at least onebioactive agent may be a DNA linking agent including Carboplatin,Cisplatin and Oxaliplatin.

In another embodiment of the present disclosure, the at least onebioactive agent may be a hormons including Anastrozole, Abiraterone,Amifostine, Bexarotene, Bicalutamide, Buserelin, Cyproterone, Degarelix,Exemestane, Flutamide, Folinic acid, Fulvestrant, Goserelin, Lanreotide,Lenalidomide, Letrozole, Leuprorelin, Medroxyprogesterone, Megestrol,Mesna, Octreotide, Stilboestrol and Tamoxifen.

Biocompatible Matrix

The kit-of-parts and the methods for treatment of a bone defectdescribed in the present invention comprise a biocompatible matrix e.g.a bone graft material comprising solid particles of natural or syntheticorigin. Also compositions of the invention may comprise a biocompatiblematrix.

In a preferred embodiment, said particles comprise or consist ofsynthetic metallic structural scaffolds and/or particles or syntheticnon-metallic scaffolds and/or particles or cancellous bone orcombinations thereof.

In another preferred embodiment said particles comprise of consist of abone graft material, e.g. a bone graft material as described below.

The presence of solid particles and/or scaffolds in the biocompatiblematrix adds mechanical stability to the bone reconstruction compositionand diminishes the risk of premature loss of mechanical stability,liquefaction and/or resorption of the bone reconstruction composition.

In some embodiments of the present disclosure, the particles of thebiocompatible matrix comprise or consist of synthetic non-metallicscaffolds and/or particles and wherein said scaffold and/or particlesare 3D-printed.

In some embodiments, the particles of the biocompatible matrix compriseor consist of synthetic metallic scaffolds and/or particles, such asspheres or scaffolds. The synthetic metallic particles may comprise orconsist of titanium or tantalum.

The particles of the biocompatible matrix preferably have a particlesize of at least 0.5 mm, such as at least 1 mm, such as at least 2 mm,such as at least 3 mm. Said particles may have any shape, they can forexample be diamonds, spheres or cubes.

Other examples of synthetic metallic structural scaffolds and/orparticles or synthetic non-metallic scaffolds and/or particles arenatural or synthetic polymers, composites (chitosan, collagen etc.ceramics, PEEK, carbon fiber reinforced PEEK, carbon) and naturalcoralline hydroxyapatite.

Bone Graft Material

In some embodiments of the present disclosure, the biocompatible matrixcomprises or consists of a bone graft material. Thus, the methods maycomprise impacting a damaged bone with the bone reconstructioncomposition and biocompatible matrix components such as said bone graftmaterial and/or synthetic particles. The bone graft material comprisesbone from a human or non-human animal. It is preferred that the bonegraft material comprises bone from the same species, as the species tobe treated with the bone graft material. Thus, frequently it ispreferred that said bone graft material comprises bone from a humanbeing.

In preferred embodiments of the invention, the bone graft materialconsists of bone from a human or non-human animal. In particular, thebone graft material may consist of human bone.

Said bone graft material may be autogenic or allogenic bone, i.e. thebone graft material may comprise or consist of bone from the individualto be treated, or it may comprise or consist of bone from anotherindividual of the same species, or a combination of both.

Therefore, in some embodiments of the present disclosure, thebiocompatible matrix, e.g. a bone graft material, comprises particles ofcancellous bone, wherein the cancellous bone is allograft and/orautograft.

For example the biocompatible matrix e.g. the bone graft material mayessentially consist of cancellous bone, i.e. at least 70%, preferably80%, such as at least 85% of the biocompatible matrix consists ofcancellous bone, for example human cancellous bone. The remainder of thebiocompatible matrix may consist of remnants of material naturallyassociated with cancellous bone, for example cortical bone, soft tissuesof bone, lipids and/or blood. In some embodiments, the cancellous bonecomprises no more than 10% water and no more than 5% lipids.

In some embodiments of the present disclosure, said bone graft materialcomprises demineralized bone matrix.

In some embodiments of the present disclosure, said bone graft materialcomprises between 1 and 10 ml of demineralized bone matrix.

In some embodiments of the present disclosure, said bone graft materialcomprises between 1 and 10 g of demineralized bone matrix.

In some embodiments of the present disclosure, said bone graft materialis allograft and/or autograft bone, for example vascularized autograft.

Frequently, said bone, e.g. cancellous bone is present in thebiocompatible matrix e.g. in the bone graft material in the form ofsmaller particles, for example in particles having a particle size of nomore than 5 mm, such as no more than 4 mm, for example no more than 3mm, for example in the range of 2 to 3 mm. In particular, the bone, e.g.cancellous bone may be present in the bone graft material in the form ofsmaller particles having a size in the range of −3.5 to −6 mesh, forexample in the range of −4 to −6 mesh, such as in the range of −5 to −6mesh. A “−” before the sieve mesh indicates the particles pass throughthe sieve. Thus, by way of example if a material is described as −6mesh, then 90% or more of the material will pass through a 6-mesh sieve.

Said bone, e.g. cancellous bone may undergo processing that may comprisefragmenting the material, cleaning the material and/or disinfecting thematerial before it is ready for use as a biocompatible matrix. Said bonein the form of particles may be prepared by milling, crushing or othermean of fragmentation. For example, the bone in the form of particlesmay be prepared using a bone mill or a device having the same function.

In a preferred embodiment, said biocompatible matrix e.g. bone graftmaterial is frozen cancellous allograft bone taken from a hospital bonebank and stored as per the routine procedure of the hospital. Suchmaterial may be devoid of cortical and soft tissue remnants.

In another embodiment, said biocompatible matrix e.g. bone graftmaterial is autograft bone, wherein said bone is cancellous autograft orcorticocancellous autograft shaped and applied into the outer surfaceareas of an individual bone defect in the area of a cortical window. Inthis embodiment, longer curing time and more compression during curingmay apply due to the higher content of blood and other liquids typicallypresent in autograft material.

In an embodiment of the present invention, said biocompatible matrixe.g. bone graft material comprises cells capable of true osteogenesis.For example when cancellous autograft and/or autograft harvested usingthe Reamer-Irrigator-Aspirator technique (RIA autograft) is used.

Curing of Composition

In a preferred embodiment of the present invention, said compositionused is cured to allow setting. Although said curing can occurspontaneously, improved interdigitation with host bone and biocompatiblematrix, e.g. bone graft material, may be achieved by keeping thematerial under compression with a gauze material after sequentialimpaction until setting is complete. The curing procedure may beperformed for few minutes and/or until complete setting.

In a preferred embodiment of the present disclosure, the bonereconstruction composition cures in a time range of 2 to 60 minutesafter all ingredients of said composition are mixed. The curing time mayvary, it can for example be 2 to 50 minutes, preferably 2 to 40 minutes,preferably 2 to 30 minutes, preferably 2 to 20 minutes, preferably 2 to10 minutes, preferably 2 to 5 minutes, preferably 2 to 3 minutes.

In a preferred embodiment of the present disclosure, the bonereconstruction composition thickens to a paste in a time range of 2 to10 minutes after all ingredients of said composition are mixed, such asin a time range of 2 to 8 minutes, such as in a time range of 2 to 7minutes, such as in a time range of 2 to 6 minutes, such as in a timerange of 2 to 5 minutes, such as in a time range of 2 to 3 minutes afterall ingredients of said composition are mixed.

Method of Producing Bone Reconstruction Composition

In an embodiment of the present invention, a composition to be usedtogether with bone graft material for treatment of bone defect isprepared. The bone reconstruction composition may be prepared by anyuseful method.

In one embodiment the bone reconstruction composition may be prepared bya method comprising the steps of:

-   -   a. providing a biocompatible carrier    -   b. providing one or more bioactive agents, e.g. an        antiresorptive agent, an antibiotics, or other agents described        in the section “Bioactive agent”    -   c. optionally providing a solvent    -   d. mixing said the biocompatible carrier with the one or more        bioactive agents and optionally with a solvent until a paste is        formed    -   e. optionally adding additional bioactive agents in solvent        followed by mixing.

In some embodiments, the bioactive agents are provided in a solvent.

In other embodiments, additional solvent is added to the mixture, saidsolvent being free from bioactive agents or comprising one or morebioactive agents.

In one embodiment the bone reconstruction composition may be prepared bya method comprising the steps of:

-   -   a. providing hydroxyapatite (HA) and calcium salt    -   b. mixing hydroxyapatite and calcium salt    -   c. providing an antiresorptive agent, e.g. a bisphosphonate        contained in a solvent    -   d. mixing said antiresorptive agent in said solvent with HA and        calcium salt until a paste is formed    -   e. optionally adding additional antiresorptive agent in solvent        followed by mixing.

Mixing of steps d. and e. may be for any suitable time, for example forin the range of 1 to 10 min. such as in the range of 2 to 3 min. Thesolvent may be any useful solvent e.g. water. Said water may compriseadditives, such as a buffer, a salt and/or other components e.g.mannitol. Thus, the solvent may be a buffered saline aqueous solution,e.g. a citrate buffered aqueous solution comprising a physiologicallevel of salt, e.g. in the range of 0-7 to 1.0%, such as approximately0.9% NaCl.

One specific example of a useful method to prepare composition comprisesthe following steps:

-   -   f. providing hydroxyapatite (HA) and calcium sulfate    -   g. putting hydroxyapatite and calcium sulfate in a ration 40:60        into a device that allows mixing    -   h. drawing up zolendric acid    -   i. adding an appropriate amount of zolendric acid into the        device for mixing, already containing HA and calcium sulfate    -   j. mixing till a paste is formed, having toothpaste consistency    -   k. after 3 minutes from the moment mixing was started, the        mixing is stopped for 10-20 seconds to allow stiffening    -   l. more zolendric acid is added and the mixing repeated    -   m. after 3 further minutes the composition reaches the desired        consistency    -   n. the composition is delivered into the bone cavity within the        next 3 minutes.

The composition may comprise at least 50% of HA and calcium sulfate. Thecomposition may also comprise at least 0.3 mg/ml of ZA, preferablyapproximately 0.55 mg/ml and up to 0.80 mg/ml, where ml refers to thefinal volume of the composition.

In a preferred embodiment of the invention, a longer setting time isallowed, so that the composition reaches a harder consistency. Theresulting mould is applied directly without the help of a syringe.

In some embodiments of the present disclosure, one or more bioactiveagents are added to the composition. Some bioactive agents are in liquidforms and are added to the composition as they are. Some bioactiveagents are not in liquid form and thus they are first mixed with asolvent and dissolved, and only after they are added to the mixture.

In the sections above “Bioactive agents”, “Bisphosphonate”,Antimicrobial agents“, “Growth promoting agents” and “ Other bioactiveagents” there are details regarding the various types of agents can bebeneficially be added to the composition.

In another embodiment of the present invention, an antibiotic is addedto said composition. The antibiotic may be mixed with a suitable solventprior to its addition to the composition. The dissolved antibiotic maybe added into the mixing device containing BGS prior to addition ofzolendric acid. The dissolved antibiotic may be added into the mixingdevice containing BGS after addition of zolendric acid. The compositionmay comprise at least 80 mg/ml, such as at least 97 mg/ml, such as atleast 110 mg/ml, and up to 175 mg/ml of said antibiotic or antibioticmixture, where ml refers to the final volume of the composition.

Another example of a useful method to prepare composition comprises thefollowing steps:

-   -   a. providing hydroxyapatite (HA) and calcium sulfate    -   b. putting hydroxyapatite and calcium sulfate in a ration 40:60        into a device that allows mixing    -   c. drawing up 1 mg zolendric acid 1 g fosfomycin in 10 ml        solvent    -   d. adding said solvent, containing zoledronic acid and        fosfomycin into the device for mixing, already containing HA and        calcium sulfate    -   e. mixing till a paste is formed, having toothpaste consistency    -   f. allowing the paste to set for 1-3 minutes until the        composition reaches the desired consistency    -   g. delivering the composition into the bone cavity within the        next 3 minutes.

In a preferred embodiment, the antibiotic added is fosfomycin. Inanother embodiment the antibiotic gentamicin or vancomycin is added.Additional anti-microbial agents may be added as described in thesection “Antibiotic”.

Method for Bone Reconstruction

The present invention describes methods for treatment of a bone defectin a subject, said treatment comprising providing a biocompatiblecarrier, providing biocompatible matrix, e.g. a bone graft material, andproviding impacting a bone defect with said biocompatible carrier andsaid biocompatible matrix in alternate layers to achieve a mechanicallystable bone reconstruction construct, as described in the detail in thesection above “Treatment of bone defects”.

As the bone reconstruction composition typically contains the bioactiveagents, it is applied over the entire surface of all relevant areas ofthe individual defect and in direct contact with the local host bone.Then, the biocompatible matrix is impacted in the bone defect applying acertain pressure. This layer is covered by impacting said compositioncomprising one or more bioactive agents over it. This second layer mayalso be covered with further biocompatible matrix. This third layer maybe further covered with said composition comprising one or morebioactive agents. The procedure is repeated until the desired level ofdefect fill is achieved. Impacting the reconstruction construct withinsaid bone defect with said composition and said biocompatible matrixduring or at the end of the reconstruction process can contribute toachieve a denser and more mechanically stable bone reconstructionconstruct.

Bone defect reconstruction may require additional mechanicalstabilization with appropriate internal or external bone fixationdevices including but not limited to standard or custom wires, pins,screws, plates, nails, prosthetic- and/or external fixation devices, butalso auto- or allograft bone with- or without vascularized bone grafts,which can help reinforcing the structure of the defected bone.

In one embodiment of the invention, said method comprises forming atleast 2 layers, one comprising the biocompatible matrix and onecomprising the bone reconstruction composition, so that the bone defectis repaired.

In a preferred embodiment, said method comprises forming at least 3layers, such as at least 4 layers, such as at least 5 layers, such as atleast 6 layers, wherein at least one comprises the biocompatible matrixand at least one comprises the composition, so that the bone defect isrepaired.

In one aspect, the present disclosure relates to a method for bonereconstruction in an individual suffering from a bone defect, saidmethod comprising performing the method as described herein, therebyobtaining growth of healthy bone on the site of the bone defect in saidindividual.

EXAMPLES Example 1 Reconstruction of Metastatic Bone Defects with aBisphosphonate Eluting Bone Graft Substitute

Six patients (5 f, 1 m, mean age 64 (range 37-81) who had undergonereconstruction of metastatic bone defects with implantation of agentamycin eluting sulfate-apatite bone graft substitute (Cerament™|G,BONESUPPORT, Lund, Sweden) which was additionally loaded with zoledronicacid, were prospectively followed for a mean of 12 months (range 6-17).In all 5 female patients, the indication for treatment was a partiallycontained bone defect associated with incipient or actual pathologicfracture of the acetabulum (n=3) or proximal humerus (n=2) secondary tometastatic breast cancer. The only male patient had wide resection of asolitary lung cancer metastasis from the proximal femur.

Sequential imaging (X-ray/CT) demonstrated progressive consolidation ofthe inserted graft material without any evidence of persistentosteolysis or local recurrence. Rapid and homogeneous remodelingtypically started in well-contained areas with cancellous bone contact.Substantial bone formation was also observed in uncontained areas wheregraft material had been applied to the surface of metallic implants orsurrounding cortical bone in some cases.

Example 2 Early Clinical Experience with Local Bisphosphonate Deliveryfor Bone Defect Reconstruction in Aggressive Benign Bone Tumors

11 patients (9 f, 2 m, mean age 35 (range 18-62)) with aggressivelybehaving benign bone tumors (5 GCT, 4 ABC, 2 UBC) who underwent tumorresection with curettage, high speed burring and subsequent bone defectreconstruction utilizing a combination of a biocompatible carrier, e.g.gentamycin eluting bone graft substitute (Cerament™|G, BONESUPPORT,Lund, Sweden) and a biocompatible matrix, e.g. cancellous allograft,with serial imaging (X-ray/CT) were prospectively followed for a mean of12 months (range 6-24).

Radiographic evidence of local bone formation and remodeling by farexceeded rates and amounts usually observed with either single componentalone. Rapid and homogeneous remodeling typically started in areas withcancellous bone contact in the periphery of the defects but was notlimited to the cavities only. Substantial periosteal bone formation wasalso observed in areas of ungrafted surrounding cortical bone. In theFIGS. 1-6 and 10, x-rays taken prior treatment, right after and fewmonths after treatment show bone formation and remodeling in 6 differentpatients. In particular, the treatment represented in FIGS. 1-6 and 10was performed by applying a biocompatible matrix, e.g. cancellousallograft, and a bone reconstruction composition, e.g. bone graftsubstitute (BGS) comprising zoledronic acid and they all show new boneformation and remodeling at 3 weeks to 6 months after treatment. FIG. 6shows also achievement of periosteal callus formation.

The treatment represented in FIG. 7, instead, was performed by applyingonly a bone reconstruction composition, e.g. the bone graft substitute(Cerament™|BVFG, BONESUPPORT, Lund, Sweden) not comprising zoledronicacid or any other bioactive agent. As shown in FIG. 7, completedisappearance of the bone graft substitute is observed 6 months afterthe treatment. The patient represented in FIG. 8 was affected by asepticloosening of an intercalary diaphyseal replacement in the right femur,treated with 2 stage revision and intermediate implantation of anantibiotic cement spacer and re-implantation of a new, cemented, silvercoated intercalary spacer and periprosthetic bone defect reconstruction.Part of the bone defect was filled using a biocompatible matrix, e.g.RIA autograft, DBM and allograft, and a biocompatible carrier comprisingCerament G with zoledronate (ZA) in layers, which resulted insubstantial new bone formation at 6 months (FIG. 8D). The remaining partof the bone defect was treated with only bone graft substitute (BGS)which showed no evidence of bone formation at 6 months (FIG. 8D, arrow).The patient represented in FIG. 9 was affected by a large simple cyst inthe left proximal femur, which was in a first instance treated bypercutaneous injection of a biocompatible carrier alone, e.g. the bonegraft substitute Cerament BVF, not comprising bioactive agents. 3 weekspost-operatively, the patient suffers a spontaneous fracture of theproximal femur though one of the BGS injection portals (FIG. 9B). Sopart of the bone graft substitute was removed and replaced with abiocompatible matrix, e.g. allograft, and bone graft substitutesupplemented with zoledronate and demineralized bone matrix (FIG. 9B),whereas the cranial two thirds appeared structurally intact and wereleft in place (FIG. 9B, arrow). Post-operative follow-up radiographsshow sound progressive consolidation and remodeling of the fracture(FIGS. 9C and D), while the non-substituted part progressively resorbedwithout any evidence bone formation in the femoral neck (FIGS. 9C and D,arrows).

These examples are only exemplary and do not disclose the true scope ofthe invention.

1.-55. (canceled)
 56. A curable, bone reconstruction compositioncomprising a biocompatible carrier and one or more bioactive agents,wherein the one or more bioactive agents comprise (a) an antiresorptiveagent comprising a bisphosphonate, and/or (b) fosfomycin.
 57. The bonereconstruction composition according to claim 56, wherein thebiocompatible carrier comprises calcium sulphate, calcium phosphateand/or hydroxyapatite.
 58. A composition comprising a) a bonereconstruction composition according to claim 56, wherein thebiocompatible carrier comprises calcium salts, hydroxyapatite, naturalpolymers, and/or synthetic polymers; wherein at least one of thebioactive agents accelerates curing of the composition and wherein atleast one of the bioactive agents decelerates curing of the compositionwherein the bioactive agent that accelerates curing of the compositionis fosfomycin wherein the bioactive agent that decelerates curing of thecomposition is a bisphosphonate, and b) a biocompatible matrixcomprising solid particles of natural origin; wherein the particles ofthe biocompatible matrix comprise or consist of bone graft materialand/or demineralised bone matrix (DBM).
 59. A kit-of-parts comprising:a) a bone reconstruction composition as defined in claim 58, and b) abiocompatible matrix as defined in claim
 58. 60. A method for treatmentof a bone defect in an individual in need thereof, which methodcomprises a) providing a curable bone reconstruction compositioncomprising a biocompatible carrier and at least one bioactive agent, b)providing a biocompatible matrix comprising solids particles and/orscaffolds of natural or synthetic origin, c) contacting the site of bonedefect in said individual with alternating layers of said compositionand said biocompatible matrix, wherein said treatment comprises applyingsaid bone reconstructing composition and said biocompatible matrix tothe bone defect by injection and/or moulding, wherein said bonereconstruction composition and said biocompatible matrix are impacted inat least 2 alternate layers, wherein at least one layer comprises saidbone reconstruction composition and at least one layer comprises saidbiocompatible matrix, thereby treating said bone defect.
 61. The methodaccording to claim 60, comprising providing a biocompatible carrier andone or more bioactive agents, wherein at least one of the bioactiveagents decelerates curing of the composition.
 62. The method accordingto claim 60, comprising providing a biocompatible carrier and one ormore bioactive agents, wherein at least one of the bioactive agentsaccelerates curing of the composition.
 63. The method according to claim60, comprising providing a biocompatible carrier and two or morebioactive agents, wherein at least one of the bioactive agentsdecelerates curing of the composition and one other bioactive agentaccelerates curing of the composition.
 64. The composition according toclaim 58, wherein said decelerating bioactive agent comprises abisphosphonate selected from a group consisting of Zoledronic acid,Pamidronic acid, Neridronic acid, Olpadronic acid, Alendronic acid,Ibandronic acid, Risedronic acid, pharmaceutically acceptable salts ofany of the aforementioned and combinations of any of the aforementioned.65. The composition according to claim 58, wherein said deceleratingbioactive agent comprises a growth or differentiation factor selectedfrom the group consisting of bone morphogenic proteins (BMPs)1,2,3,4,5,6,7,8a,8b, 10, 15; members of the TGF-superfamily;platelet-derived growth factor (PDGF); fibroblast growth factors (FGFs);insulin-like growth factors (IGFs); metalloproteinases; vascularendothelial growth factor (VEGFs A, B, C or D); angioprotein 1 and 2;and combinations thereof.
 66. The composition according to claim 58,wherein said decelerating bioactive agent comprises a BMP antagonistinhibitor selected from the group consisting of members of the DANfamily, members of the twisted gastrulation family, and noggin/chordin.67. The composition according to claim 58, wherein said deceleratingbioactive agent comprises a signaling protein selected from the groupconsisting of Wnt proteins, lymphoid enhancer-binding factor 1 (Lef1),β-catenin, parathyroid hormone-related protein (PTHrP), colonystimulating factors, Indian hedgehog homolog (IHH), Hypoxia-induciblefactor 1-alpha (HIF-α) and combinations thereof.
 68. The compositionaccording to claim 58, wherein said decelerating bioactive agentcomprises a human cell suspension comprising mesenchymal stem cellsand/or lymphocytes and/or platelet rich plasma (PRP) and/or the stromalvascular fraction of lipoaspirate of autogenic or allogenic origin. 69.The composition according to claim 58, wherein said deceleratingbioactive agent comprises a radioactive material selected from the groupconsisting of Cesium-131, Cesium-137, Cobalt-60, Iridium-192,Iodine-125, Palladium-103, Ruthenium-106, Radium-226 and combinationsthereof.
 70. The composition according to claim 58, wherein saiddecelerating bioactive agent comprises a cytokine selected from thegroup consisting of interleukin-1 (IL-1), interleukin-6 (IL-6), tumornecrosis factor alpha (TNF-alpha), receptor activator of nuclear factorkappa-B ligand (RANKL), core-binding factor alpha 1 (cbfa1), osteogenin,or SMADs proteins 1-8, osteogenic growth peptide (OGP), and combinationsthereof.
 71. The composition according to claim 58, wherein saiddecelerating bioactive agent comprises or consists of an antibioticselected from the group consisting of meropenem, gentamicin,tetracycline, vancomycin, tobramycin, gentamycin, cephalosporin and acombination thereof.
 72. The composition according to claim 58, whereinthe particles and/or scaffolds of the biocompatible matrix comprisessynthetic metallic structural scaffolds, and/or particles or syntheticnon-metallic scaffolds, and/or particles or cancellous bone, orcombinations thereof.
 73. The composition according to claim 58, whereinthe particles and/or scaffolds of the biocompatible matrix comprisessynthetic metallic or non-metallic scaffolds and/or particles, andwherein said scaffold and/or particles are 3D-printed.
 74. Thecomposition according to claim 58, wherein the biocompatible matrixcomprises particles of cancellous bone and wherein the cancellous boneis allograft and/or autograft.
 75. The composition according to claim58, wherein the biocompatible matrix comprises particles of cancellousbone and wherein said cancellous bone comprises demineralized bonematrix or fibers.
 76. The composition according to claim 58, wherein theparticles and/or scaffolds of the biocompatible matrix have a particlesize of no more than 20 mm.
 77. The composition according to claim 58,wherein the biocompatible matrix comprises particles of cancellous bone,wherein the cancellous bone is autograft and wherein said autograft isvascularized.
 78. The composition according to claim 58, wherein thebiocompatible matrix comprises particles of cancellous bone and whereinsaid cancellous bone comprises no more than 10% water and no more than5% lipids.